The cyclic-adenosine monophosphate (cAMP) second messenger signaling cascade is a key regulator of diverse cellular process such as insulin secretion and energy homeostasis (Seino and Shibasaki, 2005). In pancreatic [unreadable]-cells, intracellular cAMP is elevated following exposure to hormones, neurotransmitters, and other nutrient secretagogues, and potentiates glucose-stimulated insulin secretion. For this reason, the cAMP signaling pathway is currently being targeted by therapeutics for type 2 diabetes mellitus (Drucker and Nauck, 2006). Cyclic-AMP also promotes long-term changes in [unreadable]-cell function, growth, and survival through regulation of gene expression (Hinke et al., 2004), not all of which are completely understood. For patient safety it is important to understand the full-spectrum of targets of these therapeutics. There is a growing awareness in [unreadable]- cell biology that small non-coding RNA molecules called microRNAs play a major role in development and physiology. MicroRNAs hybridize with target genes and repress protein synthesis via an RNA interference (RNAi) mechanism (He and Hannon, 2004;Meister and Tuschl, 2004;Novina and Sharp, 2004;Siomi and Siomi, 2009). It is unclear, however, how microRNAs are regulated by cAMP in [unreadable]-cells. Preliminary evidence for this proposed project identifies a new target of cAMP, microRNA-375 (miR-375), a known inhibitor of insulin release. MiR-375 is the first microRNA shown to be targeted by cAMP in [unreadable]-cells and only the second microRNA known to be responsive to cAMP (Vo et al., 2005). The evidence supports a new model whereby cAMP stimulates glucose-stimulated insulin secretion in part by repressing this microRNA. This proposal tests the hypothesis that miR-375 is transcriptionally repressed by cAMP signaling in [unreadable]-cells and that this signaling occurs via the protein kinase A (PKA) pathway. Specific Aim 1 tests the model that miR-375 is transcriptionally repressed, and not post-transcriptionally degraded, in response to cAMP. Two experiments will be used to test for transcriptional repression, nuclear run-on assay and chromatin immunoprecipitation, and one experiment will be used to test for post-transcriptional degradation, pulse-chase RNA labeling. If the mechanism is determined to be transcriptional repression, then luciferase reporter assays will be used to identify a DNA promoter sequence(s) that mediates the repression. Specific Aim 2 tests the model that miR-375 and its gene targets are regulated by the PKA pathway, a key protein kinase that is activated by cAMP. An experiment will be conducted in pancreatic islets using the PKA-specific inhibitor, PKI, which will test whether miR-375 repression is mediated through PKA. A second experiment will test whether physiological agonists of cAMP signaling repress miR-375 and whether their effects are PKA-dependent. A third experiment will test whether miR-375 target genes are regulated by cAMP via their miR-375 binding site. Successful completion of this project should result in a model for cAMP regulation of miR-375 which could be used to design diabetes therapies that complement and enhance existing therapeutics that target the cAMP pathway. PUBLIC HEALTH RELEVANCE: The rapid rise in the numbers of cases of type 2 diabetes mellitus is creating an unsustainable burden on the health care systems of the U.S. and world, and while there is no cure for the disease, there are therapies which alleviate some of the symptoms and complications. Several therapeutics activate a signaling pathway in pancreatic [unreadable]-cells called the cyclic-adenosine monophosphate (cAMP) second messenger cascade which boosts postprandial insulin secretion. This proposal will characterize a new target of cAMP signaling, microRNA-375, which has the potential to be used as a complementary therapy to enhance the efficacy of existing therapeutics that target the cAMP signaling pathway.